title: Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease.
abstract: The I-CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double-strand breaks and enhancing DNA recombination reactions in maize cells. The DNA-binding properties of this protein were re-designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single-chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium-mediated transformation, the cleavage resulted in mutations at a co-delivered extra-chromosomal ms26-site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26-site in 5.8% of transgenic T(0) plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double-strand break repair generated by non-homologous end joining. One of 21 mutagenized T(0) plants carried two mutated alleles of the MS26 gene. As expected, the bi-allelic mutant T(0) plant and the T(1) progeny homozygous for the ms26 mutant alleles were male-sterile. This paper described the second maize chromosomal locus (liguless-1 being the first one) mutagenized by a re-designed I-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.
authors.affiliation: DuPont/Pioneer Agricultural Biotechnology, 8305 N.W. 62nd Avenue, Johnston, IA, 50131, USA.
authors.forename: Vesna
authors.initials: V
authors.lastname: Djukanovic
authors.forename: Jeff
authors.initials: J
authors.lastname: Smith
authors.forename: Keith
authors.initials: K
authors.lastname: Lowe
authors.forename: Meizhu
authors.initials: M
authors.lastname: Yang
authors.forename: Huirong
authors.initials: H
authors.lastname: Gao
authors.forename: Spencer
authors.initials: S
authors.lastname: Jones
authors.forename: Michael G
authors.initials: MG
authors.lastname: Nicholson
authors.forename: Ande
authors.initials: A
authors.lastname: West
authors.forename: Janel
authors.initials: J
authors.lastname: Lape
authors.forename: Dennis
authors.initials: D
authors.lastname: Bidney
authors.forename: Saverio
authors.initials: S
authors.lastname: Carl Falco
authors.forename: Derek
authors.initials: D
authors.lastname: Jantz
authors.forename: Leszek
authors.initials: L
authors.lastname: Alexander Lyznik
mesh_terms: D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D018390:Gene Targeting; D017343:Genes, Plant; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051479:Plant Infertility / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000502:physiology; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics* / Q000502:physiology
chemical_list: D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI
keywords: Zea mays L; double-strand break; homing endonuclease; maize; mutagenesis; technical advance